【摘要】 巴西橡胶树(Hevea brasiliensis Muell.)是重要热带经济作物,其主要经济价值来源于其树皮组织中的乳管细胞所合成的天然橡胶。研究橡胶树树皮及乳管中的蛋白质表达及功能是了解天然橡胶生物合成及调控的重要方法。然而由于橡胶树树皮中次生代谢物丰富,树皮木栓化严重,总蛋白提取难度较高,目前尚未见有成熟的橡胶树树皮总蛋白提取方法的相关报道。比较TCA/丙酮沉淀法、TCA/丙酮—双乙法和TCA/丙酮-TrisHCl法等3种橡胶树树皮总蛋白提取方法,比较其提取蛋白浓度、蛋白质提取操作步骤及试剂用量,探索出一种简单高效的橡胶树树皮蛋白质提取方法;并通过蛋白质浓度测定、聚丙烯酰胺凝胶电泳(SDS-PAGE)效果分析以及内参蛋白tubulin的Western blot分析验证提取效果。结果表明,TCA/丙酮-Tris-HCl法比传统的TCA/丙酮法提取效率更高,得到的蛋白量更多,且条带清晰,适用于SDS-PAGE,Western blot等后续蛋白分析实验。更多还原

【Abstract】 Hevea brasiliensis is an important tropical crop, and its economical value comes from the nature rubber produced in the laticifers in the bark. The proteins in the bark and laticifers are the major targets in the study of the rubber biosynthesis and regulation. However, the total protein extraction in the rubber tree bark is difficult due to the abundant metabolites and wooden cork in it. Until now, there are no reports on the extraction methods of total protein from the rubber tree bark. In this study, a comparison between three extraction methods, i. e. TCA/Acetone, TCA/Acetone-Ethanol-ether method and TCA/Acetone-Tris-HCl method were conducted. The protein concentration, extraction protocols and the reagent consumptions were compared. Finally, an easy and effective protocol for rubber tree bark protein extraction was proposed. The extraction effect was further validated by protein concentration measurement, polyacrylamide gel electrophoresis(SDS-PAGE) analysis and Western blot analysis with internal reference protein tubulin. The results showed that the TCA/acetone-Tris-HCl method is more efficient than the traditional TCA/acetone method in extracting more proteins, and the bands are clear,which is suitable for subsequent protein analysis experiments such as SDS-PAGE and Western blot.更多还原